Ilex guayusa Loes., known as guayusa, is a plant that has been used for generations by the indigenous peoples of the Ecuadorian Amazon for its medicinal properties, notably its stimulating effects. This bioactivity is primarily due to the presence of xanthine alkaloids such as caffeine, theobromine, and theophylline. In this study, the concentration of each alkaloid was determined in thirty populations throughout the Amazon region, with data ranging from 2065.0 mg/100 g to 364.8 mg/100 g for caffeine; 225.8 mg/100 g and 74.6 mg/100 g for theobromine; and 4.9 mg/100 g and 1.5 mg/100 g for theophylline. A statistical analysis was conducted on twenty-four guayusa populations by integrating alkaloid concentrations with altitude, climatic, and soil variables. Caffeine showed high variability among populations and was only weakly associated with elevation, whereas theobromine and theophylline were determined primarily by climatic and soil factors. These results indicate that methylxanthine accumulation in Ilex guayusa is driven by the combined influence of climatic and edaphic conditions rather than by a single environmental factor.